Several animal models of cerebral ischemia have been developed to simulate the human condition of stroke. This protocol describes the endothelin-1 (ET-1) induced middle cerebral artery occlusion (MCAO) model for ischemic stroke in rats. In addition, important considerations, advantages, and shortcomings of this model are discussed.
Stroke is the number one cause of disability and third leading cause of death in the world, costing an estimated $70 billion in the United States in 20091, 2. Several models of cerebral ischemia have been developed to mimic the human condition of stroke. It has been suggested that up to 80% of all strokes result from ischemic damage in the middle cerebral artery (MCA) area3. In the early 1990s, endothelin-1 (ET-1) 4 was used to induce ischemia by applying it directly adjacent to the surface of the MCA after craniotomy. Later, this model was modified 5 by using a stereotaxic injection of ET-1 adjacent to the MCA to produce focal cerebral ischemia. The main advantages of this model include the ability to perform the procedure quickly, the ability to control artery constriction by altering the dose of ET-1 delivered, no need to manipulate the extracranial vessels supplying blood to the brain as well as gradual reperfusion rates that more closely mimics the reperfusion in humans5-7. On the other hand, the ET-1 model has disadvantages that include the need for a craniotomy, as well as higher variability in stroke volume8. This variability can be reduced with the use of laser Doppler flowmetry (LDF) to verify cerebral ischemia during ET-1 infusion. Factors that affect stroke variability include precision of infusion and the batch of the ET-1 used6. Another important consideration is that although reperfusion is a common occurrence in human stroke, the duration of occlusion for ET-1 induced MCAO may not closely mimic that of human stroke where many patients have partial reperfusion over a period of hours to days following occlusion9, 10. This protocol will describe in detail the ET-1 induced MCAO model for ischemic stroke in rats. It will also draw attention to special considerations and potential drawbacks throughout the procedure.
This protocol was approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Florida and is in compliance with the “Guide for the Care and Use of Laboratory Animals” (eighth edition, National Academy of Sciences, 2011).
Materials
1. Pre-surgical Steps
2. Surgical Steps
At this point, a guide cannula can be inserted (step 7) or a direct ET-1 injection through the bur hole can be performed (proceed directly to step 16).
After these steps, the surgical incision can be closed and the cannula dummy can be screwed into the guide cannula. Alternatively, ET-1 induced MCAO can be performed on the rat after a period of recovery from the cannula implantation surgery. For this method, step 19 should be performed next and steps 14-18 can be performed at a later time. To perform guide cannula implantation and ET-1 injection during the same surgery, step 14 should be performed next.
1. Post-Op neurological evaluation
After the animal regains consciousness, a wide variety of tests can be used to evaluate neurological deficits including balance, grip strength, paw placing, postural asymmetry and staircase climbing. The sunflower seed task is a gross assessment of motor and sensory function that has significant correlation with infarct volume7, 12. During this task, rats are timed while opening and consuming 5 sunflower seeds. The five seeds are placed in one corner of an empty, dry, plastic cage and the time spent manipulating the seeds is recorded. In addition, the number of shell pieces in the cage after all seeds have been opened and consumed is recorded. Rats with greater neurological deficit will take longer to open and consume the seeds and will break the shells into more pieces. Although not a formal part of this task, it is easy to observe frequent mishandling and dropping of the sunflower seeds by rats after experimental stroke. This includes the use of primarily one forepaw, and ineffective biting of the shell during attempts to open.
2. Staining and quantitative measurement of brain infarct volume
After occlusion and reperfusion of the MCA to establish transient cerebral ischemia, animals are euthanized and their coronally sectioned brains are stained with 2,3,5-triphenyltetrazolium chloride (TTC) to evaluate the infarct volume (Figure 2) as discussed in our previous publications7, 13.
Figure 1. Dorsal view of the rat skull depicting hardware placement. This dorsal view of the rat skull with anterior oriented to the left shows the location of bregma and lambda with respect to the sutures that can be visualized after clearing connective tissue from the skull surface. The lateral skull ridge, guide cannula, LDF probe holder, and anchoring screws are also shown on the diagram with relative relationships to skull land marks and each other.
Figure 2. TTC-stained serial coronal brain sections (2 mm) from rat subjected to ET-1 induced MCAO. A representative brain from a rat after ET-1 induced MCAO is shown. This brain was removed and rinsed in ice cold PBS prior to being sectioned coronally in 2 mm thick sections starting just caudal to the olfactory bulbs. The slices were then incubated in 0.05% TTC diluted in PBS at 37 °C prior to being quenched in PBS and then fixed briefly in formalin. Tissue stained red after exposure to TTC represents viable gray matter. The viable gray matter in the hemisphere ipsilateral to ET-1 injection can be compared to the gray matter of the contralateral hemisphere to calculate the area of the cerebral infarct.
The ET-1 induced MCAO is an established model of experimental ischemic stroke that is regularly used in multiple rat strains. Many variables, such as rat strain, animal age, body temperature, anesthesia method, and operator expertise can lead to increased variability in infarct volumes when using this model5, 14. However, several investigators have shown that advantages of this model include the relatively non-invasive approach, dose response of cerebral blood flow to ET-1, and ability to avoid anesthesia altogether by infusion of ET-1 in conscious rats14, 15. It is notable that there are many differences in rat strain and age used throughout the literature and it is not surprising that different stereotaxic coordinates are reported depending on these variables. The coordinates used in our protocol are optimized for 8 week old male Sprague Dawley rats. Any deviation from this strain, sex, or age will likely require intracerebral injection of dye followed by necropsy to identify valid coordinates for this procedure. Additionally, ET-1 induced reduction in cerebral blood flow is dose dependent. The dose use in this method causes an infarction of approximately 30-40% of the gray matter in the hemisphere ipsilateral to the injection with a standard error of the mean of about 4-5% within an experiment7, 16. Direct visualization of proximal portions of the MCA reveals a luminal diameter that goes to 0% of baseline within 3 min and returns to baseline at approximately 30 min7. Using LDF, maximum flow was shown to decrease to approximately 10% of baseline flow in 5-7 min with gradual return to 60% of baseline after 1 hr which monitoring was discontinued16.
Other specific technical details that warrant mention are listed below:
The authors have nothing to disclose.
This work was supported by grants from the American Heart Association Greater Southeast Affiliate (09GRNT2060421), the American Medical Association, and from the University of Florida Clinical and Translational Science Institute. Adam Mecca is a NIH/NINDS, NRSA predoctoral fellow (F30 NS-060335). Robert Regenhardt received predoctoral fellowship support from the University of Florida Multidisciplinary Training Program in Hypertension (T32 HL-083810).
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